The purpose of this study is to identify the mechanism of transformation by human cytomegalovirus (HCMV) mtrII and mtrIII DNA sequences. The mtrII and mtrIII segments of HCMV strain Towne can neoplastically transform rodent cells. The mtrII (980 bp) region contains three open reading frames (ORF) of 79, 83 and 34 amino acids. We primarily focused on the role of mtrII ORFs in transformation and generated stable mtrII transformed cell lines with neomycin resistant marker by DNA transfection into NIH3T3 cells. These cell lines produced tumors in mice. Northern blot analysis indicated the presence of mtrII specific RNAs in these transformants. Using chloramphenicol acetyl transferase (CAT) assays in Cos-7 cells or CV-1 cells, we detected promoter activity in the upstream 285 bp region of mtrII when linked to CAT gene in the sense orientation with respect to ORFs.However, the entire 980 bp mtrII region had no detectable promoter activity regardless of the orientation.The 285 bp region had also weak transcriptional enhancer activity only in the sense orientation. No transactivation of the promoter sequence was detected by HIV TAT gene or CMV IE genes.These studies established that DNA sequences in mtrII can cisactivate gene expression and are in a location to regulate the expression of the mtrII ORFs. S1 nuclease analysis of total RNA using entire mtrII 980 bp sequence as probe identified several distinct RNA species.The major transcript, being protected by the entire 980 bp sequence, seems to be a hybrid RNA with a cellular gene. Two other transcripts of approximately 490 and 450 nucleotides were detected. Further studies are necessary to map these RNAs and to assess the role of the individual ORFs in mtrII mediated transformation. Regarding mtrIII (7.6 kb), the transforming activity was detected in the right hand Pst1/Xbal 3.6 kb fragment. We made deletion clones of this 3.6 kb DNA using a Stratagene exonuclease III system to determine the minimal transforming region. The project is still active.